Apoptotic entities for use in treatment of neurodegenerative and other neurological disorders

ABSTRACT

Treatment and/or prophylaxis, in mammalian patients, of neurodegenerative and other neurological medical disorders is effected by administering to the patient effective amounts of apoptotic bodies and/or apoptotic cells, preferably those derived from the patient&#39;s own white blood cells, e.g. by extracorporeal treatment of the patient&#39;s blood cells to induce apoptosis and administration of the apoptotic bodies and/or cells so formed to the patient.

FIELD OF THE INVENTION

This invention relates to biochemical and biological compositions and tothe uses thereof in the treatment and/or prophylaxis of variousneurodegenerative and other neurological disorders in mammalianpatients. More particularly, it relates to treatment and prophylaxis ofneurodegenerative and other neurological disorders by administration ofcompositions containing the mammalian cellular materials and fragmentsthereof, and to the compositions containing the mammalian cellularmaterials and fragments themselves, and to processes for preparing suchcompositions.

BACKGROUND OF THE INVENTION

Two mechanisms of cell death in the body are recognized, necrosis andapoptosis. Apoptosis is the process of programmed cell death, describedby Kerr et al in 1992 [Kerr J F R, Wyllie A H, Currie A R (1992).“Apoptosis: a basic biological phenomenon with wide-ranging implicationsin tissue kinetics. “British Journal of Cancer 26: 239-257”], by whichsteady-state levels of the various organ systems and tissues in the bodyare maintained as continuous cell division and differentiation takesplace. Cells undergoing apoptosis often exhibit distinctivemorphological changes such as a pronounced decrease in cell volume,modification of the cytoskeletons resulting in pronounced membraneblebbing, a condensation of the chromatin, and degradation of the DNAinto oligonucleosomal fragments. Following these morphological changes,an apoptotic cell may break up into a number of small fragments known asapoptotic bodies, consisting essentially of membrane-bound bodiescontaining intact organelles, chromatin etc. Apoptotic bodies arenormally rapidly removed from the body by phagocytosis by macrophages,dendritic cells and other antigen presenting cells, before they canbecome lysed and release their potentially pro-inflammatoryintracellular contents.

In simple outline, apoptosis is thought to proceed as follows. Threephases can be identified in the apoptotic mechanism of programmed celldeath:

Induction phase;

Effector phase; and

Degradation phase.

The induction phase is dependent, in part, on specific interactions ofdeath-inducing signals at the cell surface membrane. One common signalis initiated by the binding of specific ligands to receptors of the TNFreceptor family present on the cell membrane. One important suchreceptor is Fas (APO-1, CD95), which interacts with Fas-ligand toinitiate apoptosis.

The effector phase, activated by the binding of receptors and ligands ofthe induction phase, leads to the activation of caspases,cystinyl-aspartate-requiring proteinases (proteolytic enzymes),including caspases 1 and 8. This activation may be associated with achange in the permeability of mitochondria, allowing the release ofcytochrome-c which is involved in caspase activation. Activated caspasesinitiate a chain of lethal proteolytic events culminating in the changesin chromatin and cytoskeletal components seen in apoptosis.

Many cells undergoing apoptosis can be identified by a characteristic‘laddering’ of DNA seen on agarose gel electrophoresis, resulting fromcleavage of DNA into a series of fragments. These changes occur a fewhours before death of the cell as defined by the ability of a cell toexclude vital dyes. The appearance of DNA laddering on agarose gelelectrophoresis following extraction of DNA from cells is one recognisedmethod of identification of apoptosis in cells [Loo, D. T. and Rillema,J. R. (1998) “Measurement of Cell Death,” Methods in Cell Biology 57:251-264], although it is not always sensitive enough to detectapoptosis. In situ labelling of nuclear DNA fragmentation, for example,using commercially available terminal dUTP nick end labelling (TU-NEL)assays, are an alternative and more reproducible measure for thedetermination of fragmented DNA in apoptotic cells and cells undergoingapoptosis [Gavrieli Y, Sherman Y, Ben-Sasson S A (1992)“, Identificationof programmed cell death in situ via specific labelling of nuclear DNAfragmentation”. Journal of Cell Biology 119: 493-501].

During apoptosis, phosphatidylserine becomes exposed externally on thecell membrane [Fadok V A, Voelker D R, Campbell P A, Cohen J J, BrattonD L, Henson P M (1992), “Exposure of phosphatidylserine on the surfaceof apoptotic lymphocytes triggers specific recognition and removal bymacrophages”. Journal of Immunology 148: 2207-2216] and this exposedphosphatidylserine binds to specific receptors to mediate the uptake andclearance of apoptotic cells in mammals [Fadok V A, Bratton D L, Rose DM, Pearson A, Ezekewitz R A B, Henson P M (2000), “A receptor forphosphatidylserine-specific clearance of apoptotic cells”, Nature 405:85-90]. The surface expression of phosphatidylserine on cells is anotherrecognised method of identification of apoptotic cells.

Changes in mitochondrial integrity are intimately associated withapoptosis, resulting in alterations in mitochondrial membranepermeability and the release of cytochrome-c from the mitochondria intothe cell cytoplasm [Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I,Brenner, C., Larochette, N., Prevost, M. C., Alzari, P. M. and Kroemer,G. (1999) “Mitochondrial Release of Caspase-2 and -9 during theApoptotic Process”, Journal of Experimental Medicine, 189: 381-394].Measurement of changes in mitochondrial membrane potential, reflectingchanges in mitochondrial membrane permeability, is another recognisedmethod of identification of apoptotic cells.

A number of other methods of identification of cells undergoingapoptosis and of apoptotic cells, many using monoclonal antibodiesagainst specific markers for apoptotic cells, have also been describedin the scientific literature.

Necrosis, in contrast, is cell death of a pathological nature, resultingfrom injury, bacterial toxin effects, inflammatory mediators, etc., andinvolving membrane rupture and release of intracellular contents to thesurrounding tissue, often with harmful inflammatory consequences.Accordingly, one of the ways in which necrotic cells may be detected andcharacterized is by detection of compromised cell membranes, e.g. bymethods of staining with propidium iodide followed by flow cytometry ormicroscopy.

SUMMARY OF THE INVENTION

This invention is directed, in part, to the novel and unexpecteddiscovery that administration to a mammal of apoptotic cells and/orapoptotic bodies previously prepared ex vivo, can be used in theprophylaxis and/or treatment of neurodegenerative and/or otherneurological disorders in the treated mammal.

Accordingly, in one of its composition aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient and an effective amount of apoptotic cells and/orapoptotic bodies.

The pharmaceutical compositions preferably employ an aqueous basedpharmaceutically acceptable excipient although other excipients can beused.

As noted above, these compositions are useful in the prophylaxis and/ortreatment of neurodegenerative and/or other neurological disorders inmammals. Accordingly, in one of its method aspects, this invention isdirected to a method for the treatment of or prophylaxis againstneurodegenerative and other neurological medical disorders in amammalian patient, which comprises administering to the patient aneffective amount of apoptotic bodies and/or apoptotic cells.

These methods are preferably accomplished by administering to thepatient the pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE is a graph showing a comparison of net ear swelling in micetreated with the compositions of this invention and a control group.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention is directed to the treatment and/or prophylaxis ofneurodegenerative and/or other neurological disorders by theadministration of apoptotic cells and/or bodies.

Neurodegenerative disorders, including Down's syndrome, Alzheimer'sdisease and Parkinson's disease, are associated with increased levels ofreactive oxygen species (ROS), certain inflammatory cytokines, includinginterleukin-1β (IL-1β) [see Griffin W S T, Stanley L C, Ling C, White L,Macleod V. Perrot L J, White C L, Araoz C (1989). Brain interleukin 1and S-100 immunoreactivity are elevated in Down's syndrome and Alzheimerdisease. Proceedings of the National Academy of Sciences USA867611-7615; Mogi M, Harada M, Narabayashi H, Inagaki H, Minami M,Nagatsu T (1996). Interleukin (IL)-1 beta, IL-1, IL-4, IL-6 andtransforming growth factor-alpha levels are elevated in ventricularcerebrospinal fluid in juvenile parkinsonism and Parkinson's disease.Neuroscience Letters 211:13-16]. It has also been shown that IL-1inhibits long-term potentiation in the hippocampus [Murray C A, Lynch MA (1998). Evidence that increase hippocampal expression of the cytokineinterleukin-1β is a common trigger for age and stress-inducedimpairments in long-term potentiation. Journal of Neuroscience18:2974-2981]. Long-term potentiation in the hippocampus is a form ofsynaptic plasticity and is generally considered to be an appropriatemodel for memory and learning [Bliss T V P, Collinridge G L, (1993). Asynaptic model of memory: long-term potentiation in the hippocampus,Nature 361:31-39]. Thus, inappropriate cytokine expression in the brainis currently believed to be involved in the development and progressionof neuroinflammatory diseases.

Neurodegenerative and other neurological disorders treatable by thepresent invention include Down's syndrome, Alzheimer's disease,Parkinson's disease, senile dementia, depression and the like. Insummary, it can be substantially any neurodegenerative or otherneurological disorder.

“Apoptotic cells” and “apoptotic bodies,” as the terms are used herein,means cells and cell bodies which exhibit one or more of the followingapoptosis-characterizing features: surface exposure ofphosphatidylserine, as detected by standard, accepted methods ofdetection such as Annexin V staining; alterations in mitochondrialmembrane permeability measured by standard, accepted methods (e.g.Salvioli, S., Ardizzoni, A., Franceschi, C. Cossarizza, A. (1997) “JC-1,but not DiOC6(3) or Rhodamine 123, is a Reliable Fluorescent Probe toassess Delta Psi Changes in Intact Cells: Implications for Studies onMitochondrial Functionality during Apoptosis,” FEBS Letters 411: 77-82];evidence of DNA fragmentation such as the appearance of DNA laddering onagarose gel electrophoresis following extraction of DNA from the cells[Teiger, E., Dam, T. V., Richard, L., Wisnewsky, C., Tea, B. S.,Gaboury, L., Tremblay, J., Schwartz, K. and Hamet, P. (1996) “Apoptosisin Pressure Overload-induced Heart Hypertrophy in the Rat,” Journal ofClinical Investigation 97; 2891-2897], or by in situ labeling (seeGavrieli et al., 1992, referenced above).

The apoptotic cells and/or apoptotic bodies for use in the presentinvention preferably comprise not more than about 35 weight percent ofnecrotic cells and/or necrotic bodies based on the total weight of theapoptotic cells/bodies and necrotic cells/bodies; more preferably, notmore than about 20 weight percent; and even more preferably, not morethan about 10 weight percent. At these levels, the presence of suchnecrotic cells and/or bodies are believed not to significantly alter invivo processes. In its most preferred embodiment, the apoptoticcells/bodies are substantially free of necrotic cells and or bodies(i.e., less than about 2 weight percent of necrotic cells/bodies).

The apoptotic cells and/or apoptotic bodies for use in the presentinvention are prepared ex vivo from mammalian cells that are compatiblewith those of the mammalian patient. They can be prepared fromsubstantially any type of mammalian cell including cultured cell lines.Preferably they are prepared from a cell type derived from the mammalianpatient's own body or from an established cell line. More preferablythey are prepared from white blood cells of blood compatible with thatof the mammalian patient, more preferably from the patient's own whiteblood cell and even more preferably from the patient's own Tlymphocytes. Even more preferably they are prepared from an establishedcell line. The apoptotic cells and/or apoptotic bodies are preparedextracorporeally prior to administration to the patient. Thus, in oneembodiment, an aliquot of the patient's blood may be withdrawn, e.g. byvenipuncture, and at least a portion of the white cells thereofsubjected extracorporeally to apoptosis inducing conditions.

A variety of methods of inducing apoptosis in mammalian cells, so as tocreate apoptotic cells and apoptotic bodies, are known in the art andessentially any of these can be adopted in preparing apoptotic bodiesfor use in the present invention. One such method is the subjection ofthe cells to ionizing radiation (γ-rays, x-rays, etc.) and/ornon-ionizing electromagnetic radiation including ultraviolet light.Apoptosis can be induced by subjecting cells to ultrasound.

Another method is the treatment of the cells with drugs such asnon-specific protein kinase inhibitors as exemplified by staurosporine(see Bombeli, Karsan, Tait and Hirlan, (1997) “Apoptotic VascularEndothelial Cells Become Procoagulant”, Blood, Vol. 89:2429-2442). Also,certain chemotherapeutic agents used for the treatment of malignanttumours induce apoptosis, for example adriamycin, as can statin drugs(3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) [GuijarroC, Blanco-Colio L M, Ortego M, Alonso C, Ortiz A, Plaza J J, Diaz C,Hernandez G, Edigo J (1998), “3-hydroxy-3-methylglutaryl coenzyme Areductase and isoprenylation inhibitors induce apoptosis of vascularsmooth muscle in culture, Circulation Research 83: 490-500] andcolcicine [Suzuki Y (1998)”, “Cell death, phagocytosis and neurogenesisin mouse olfactory epithelium and vomeronasal organ after colcicinetreatment,” Annals of the New York Academy of Sciences 855: 252-254].The use of ligands for death receptors on cells, such as Fas-ligand,will be apparent for inducing apoptosis from the discussion of apoptosisabove.

Yet another method is the application of oxidative stress to cellsextracorporeally (see for example Buttke and Sandstrom (1994) “OxidativeStress as a Mediator of Apoptosis,” Immunology Today, Vol. 15:7-10).This can be achieved by treating the cells, in suspension, with chemicaloxidizing agents such as hydrogen peroxide, other peroxides andhydroperoxides, ozone, permanganates, periodates, and the like.Biologically acceptable such oxidizing agents are preferably used, so asto reduce potential problems associated with residues and contaminationsof the apoptotic cells and apoptotic bodies so formed.

The present invention is not restricted to any particular method ofproducing apoptotic cells and apoptotic bodies, for use herein, and anysuitable, known process can be used.

Methods for the detection and quantitation of apoptosis can be used todetermine the presence and level of apoptosis in the preparation to beadministered to the patient in the present invention. At least one ofthe methods from those described in the introduction above should beused to confirm the level of apoptosis achieved prior to administration.They are suitably purified prior to use, by methods known in the art,such as differential centrifugation.

In preparing the apoptotic cells and/or apoptotic bodies, care should betaken not to apply excessive levels of oxidative stress, radiation, drugtreatment, etc., since otherwise there is a significant risk of causingnecrosis of at least some of the cells under treatment. Necrosis causescell membrane rupture and the release of cellular contents often withbiologically harmful results, particularly inflammatory events, so thatthe presence of necrotic cells and their components along with theapoptotic bodies is best avoided. Appropriate levels of treatment of thecells to create apoptotic bodies for use in the present invention dependto some extent on the nature of the chosen cells and cellularcomposition, and the type of treatment chosen to induce apoptosis. Suchappropriate levels are readily determinable by those skilled in the art,having regard to the available scientific literature on the subjectincluding the above-reference articles.

One preferred process according to the present invention involves theculture of cells from the patient, or a compatible mammalian cell line.The cultured cells may then be treated to induce apoptosis and to createapoptotic cells and/or apoptotic bodies therein. The cells, suspended inthe patient's plasma or another suitable suspension medium, such assaline or a balanced mammalian cell culture medium, can then beadministered as indicated below. The numbers of apoptotic cells and/orbodies can be determined by published methods available in thescientific literature on the subject including the above-referencearticles. The numbers of such apoptotic cells and/or apoptotic bodiesrequired for administration to the patient to obtain the requiredclinical benefit will vary depending on the source of cells, thepatient's condition, the age and weight of the patient and otherrelevant factors which are readily determinable by the attendingclinician.

Thus, an example of a preferred process according to the presentinvention accordingly involves extraction of an aliquot of blood fromthe patient to be treated, separation of the white cells therefrom, andtreatment of the white cells under apoptosis-causing conditions, so asto create a cellular composition in which significant numbers of thewhite cells therein have been apoptosed so as to create thereinsubstantial numbers of apoptotic cells or bodies. Then the treatedcomposition is administered to the patient. More preferably, Tlymphocytes, isolated from the blood by known means, and suspended asabove, may be used as a source of apoptotic cells and apoptotic bodies.

The number of viable cells selected for treatment to create apoptoticcells and/or apoptotic bodies is suitably up to about 4×10⁹, preferablyfrom about 1,000,000 to about 1,000,000,000 and most preferably fromabout 50,000,000 to about 150,000,000, for each administration to ahuman patient. From about 10% to 90%, preferably from about 30% to 70%of the cellular composition for administration is comprised of apoptoticbodies and/or apoptotic cells, the balance being viable cells andnecrotic cells. Accordingly, the preferred amounts of apoptotic cellsand/or apoptotic bodies for administration are those produced bysubjecting these numbers of cells to the apoptosing conditions. Whenwhole blood is used as the source of the cells to be subjected to theapoptosis inducing conditions, these numbers of white cells areobtainable in blood aliquots of volume up to about 400 ml, preferably upto 100 ml. More specifically, 50,000,000-150,000,000 cells is equivalentto the white cells in blood aliquots of volume 10-30 ml.

The volume of the aliquot of blood withdrawn from the patient fortreatment to create apoptotic cells and/or apoptotic bodies therein issuitable up to about 400 ml, preferably from about 0.1 to about 100 mland most preferably from about 5 to about 15 ml. Accordingly, thepreferred amounts of apoptotic cells and/or apoptotic bodies foradministration are those corresponding to the numbers derivable from thewhite blood cells, or isolated T lymphocytes, contained in suchquantities of whole blood, following subjection to apoptosis-inducingconditions.

The suspension of treated apoptotic cells and/or bodies foradministration to the patient is prepared in a biologically acceptableliquid suspending medium, such as the patient's serum or plasma, salineor balanced mammalian cell culture medium. The addition of otherfactors, such as cytokines, hormones, products of stressed cells orother appropriate biologically active material may enhance the benefitof the administered apoptotic cellular materials. The aliquot can beintroduced into the patient's body by any suitable method, mostpreferably intramuscular injection but also including subcutaneousinjection, mini-grafting, intra peritoneal injection, intra-arterialinjection, intravenous injection and oral administration. The apoptoticentities can be delivered to the specific body organ and/or site byusing any appropriate, known delivery system.

The compositions of this invention may optionally include apharmaceutically acceptable excipient. Some examples of suitableexcipients include sterile water, sterile saline, phosphate bufferedsaline, and the like.

When administered, the pharmaceutical compositions comprise an effectiveamount of apoptotic bodies/cells to induce a suitable prophylacticand/or therapeutic response in the patient at risk of suffering orsuffering from a neurodegenerative disease. Preferably, the compositionadministered to the mammalian patient comprises from about 10,000 to10,000,000 apoptotic cells or bodies per kilogram of body weight, morepreferably from about 500,000 to 5,000,000 and most preferably fromabout 1,500,000 to about 4,000,000 apoptotic cells and/or bodies per kgbody weight. The specific dose employed will, of course, be dependentupon the age, weight and severity of the disease in the treated patientall of which are within the skill of the attending clinician.

For most effective treatment and/or prophylaxis of mammalian disordersinvolving a neurodegenerative or neurological disorder, the patient maybe given a course of treatments with apoptotic cells and/or bodiesaccording to the invention. Each course of treatment may involveadministration to the patient of from 1 to 6 aliquots of suspendedcellular material, as described above. No more than one such aliquotshould be administered per day, and the maximum rest period between anytwo consecutive administrations should be not greater than about 21days. Booster treatments as described below may advantageously be used.To maintain the desired effects, the patient may undergo boostertreatments, with a further course of administration of aliquots ofsuspended apoptotic cells and/or apoptotic bodies as described above, atintervals of three to four months.

As noted, the present invention is applicable to the treatment andprophylaxis of a wide variety of mammalian neurodegenerative and otherneurological disorders. These include, but are not limited to, Down'sSyndrome, Alzheimer's disease, Parkinson's disease, senile dementia,depression, multiple sclerosis, Huntington's disease, peripheralneuropathies, spinal cord diseases, neuropathic joint diseases, chronicinflammatory demyelinating disease (CIPD), neuropathies includingmononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy,cystic fibrosis, neuromuscular junction disorders and myasthenias. Insummary, it can be substantially any neurodegenerative or otherneurological disorder.

The invention is further described, for illustrative purposes, in thefollowing specific examples.

EXAMPLE 1

Experiments to demonstrate the invention were conducted on laboratorymice, under approved conditions for conducting such experiments.

The effectiveness of the treatment according to a preferred embodimentof the present invention, on contact hypersensitivity (CHS), an exampleof a Th-1-cell inflammatory disorder which is known to be mediated byinflammatory cytokines, was assessed on laboratory mice, according toapproved animal experimentation procedures, using the method describedby Kondo et. al., “Lymphocyte function associated antigen-1 (LFA-1) isrequired for maximum elicitation of allergic contact dematitis” Br. J.Dermatol. 131:354-359 (1994), with minor variations. The disclosurethereof is incorporated herein by reference. Briefly, to induce CHS, theabdominal skin of each mouse was shaved and painted withdinitrodifluorobenzene DNFB, the sensitizing chemical, using 25 μl of0.5% DNFB in 4:1 acetone:olive oil solution. This sensitization wasapplied to two groups of Balb/c mice, 10 animals in total.

Apoptotic bodies were prepared from murine fibroblasts. The murinefibroblasts were treated with 50 mM sodium butyrate in RPMI medium, atconfluency for one day, and then the sodium butyrate medium was changed.To increase the number of apoptotic cells and bodies, the cells canadditionally be irradiated with UV-light (e.g. 75 mj). Supernatantcontaining floating cells is removed 24 hours following irradiation.

Apoptotic bodies were quantitated by centrifuging the supernatant (1200rpm, 5 minutes), aspirating the supernatant, washing the resulting cellpellet with PBS and centrifuging again, as above. The pellet containingthe apoptotic bodies was re-suspended in PBS. The cells were stored inPBS at 4° C. for the duration of the experiment. The cells to be stainedfor quantitation were re-suspended in 1× binding buffer at aconcentration of 1×10⁶ cells/ml. 100 μl of the cells were transferred toa 5 ml tube, and 10 μl of fluorescein-conjugated annexin V and 10 μlpropidium iodide reagent was added. The cells were gently vortexed andthe cell mixture incubated for 15 minutes at 25° C. in the dark.Following the incubation, 400 μl of 1× binding buffer is added to eachtube. The sample was analyzed on a flow cytometer over one hour.

Of the two groups of sensitized mice, the first, control group A,received no treatment. The second, test group B, was treated with aninjection of suspended apoptotic bodies prepared as described above, 50μl volume containing at least 150,000 bodies per injection of bloodsubjected to stressors as described above. Treatments, each involvingintramuscular injection of 50 μl of the respective liquid, started onthe day of sensitization, and were repeated every day for a total of sixdays. On the same day as the last treatment, but after itsadministration, the animals were challenged with DNFB, by applying tothe right ear of each animal 10 μl of 0.2% solution of DNFB in acetoneand olive oil. To the left ear of each animal was applied theacetone/olive oil solvent, without DNFB. Inflammation due to CHSmanifests itself in a swelling of the right ears. Ear thickness wasmeasured, 24 hours after challenge, with a Peacock spring-loadedmicrometer (Ozaki Co., Tokyo, Japan). The results were expressed as thethickness and difference in thickness of the right ears and the leftears of each animal, at 24 hours after challenge.

The experiments were repeated, using more sets of two groups of animals,a sufficient number of times to ensure statistical significance in theresults. A notable and significant reduction in ear thickness(inflammation) was observed with the animals treated with the apoptoticcells and apoptotic bodies suspension in accordance with the invention,as compared with the untreated group, demonstrating a significantreduction in inflammation. The results are presented in the followingTable, and on the accompanying FIGURE, as a bar graph of net earswelling (difference between right ear and left ear thickness), for eachgroup, with “standard deviation” shown by the vertical line at the topof each column.

TABLE 1 Group Left ear Right ear Difference A 17 31 14 A 18 39 21 A 1730 13 A 18 32 14 A 18 31 13 Mean: 15 S.D: 3.391165 B 21 31 10 B 18 18 0B 17 30 13 B 20 24 4 B 18 22 4 Mean: 6.2 S.D.: 5.215362

An analysis of the suspension of apoptotic cells and bodies administeredto the animals of test group B indicated the presence therein ofapproximately 40% apoptotic cells and bodies, balance viable cells andminor amounts of necrotic cells (not more than 20%), the presence ofwhich is believed to be insignificant in the in vivo process.

EXAMPLE 2

The above test procedure was repeated on similar groups of animals, acontrol group and a test group, but using a suspension of apoptoticcells and bodies on the test group which comprised about 60% apoptoticcells and bodies, balance viable cells and a minor amount (not more than20%) of necrotic cells. Essentially similar results were obtained.

The effectiveness of the processes and compositions of the presentinvention in preventing and alleviating inflammation due to CHSindicates that administration of apoptotic cells and bodies as describedup-regulates the in vivo generation of anti-inflammatory Th-2 derivedcytokines such as IL-10 (known to be implicated in CHS—see Kondo,McKenzie and Sauder, “The Journal of Investigative Dermatology,” Vol.103, 1994, page 811-814) and/or down-regulates Th-1 inflammatorycytokines such as TNFα and IL-6. These inflammatory cytokines areimplicated in inflammation-related disorders of the brain, namely theneuroinflammatory, neurodegenerative and neurological disorders such asAlzheimer's disease, senile dementia, multiple sclerosis, depression,Down's syndrome, Huntington's disease, peripheral neuropathies, spinalcord diseases, neuropathic joint diseases, chronic inflammatorydemyelinating disease (CIPD), neuropathies including mononeuropathy,polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis,neuromuscular junction disorders, myasthenias and Parkinson's disease.

Neurodegenerative diseases, including Down's syndrome, Alzheimer'sdisease and Parkinson's disease, are associated with increased levels ofcertain inflammatory cytokines, including interleukin-1β (IL-1β) [seeGriffin W S T, Stanley L C, Ling C, White L, Macleod V. Perrot L J,White C L, Araoz C (1989). Brain interleukin 1 and S-100immunoreactivity are elevated in Down syndrome and Alzheimer disease.Proceedings of the National Academy of Sciences USA 867611-7615; Mogi M,Harada M, Narabayashi H, Inagaki H, Minami M, Nagatsu T (1996).Interleukin (IL)-1 beta, IL-1, IL-4, IL-6 and transforming growthfactor-alpha levels are elevated in ventricular cerebrospinal fluid injuvenile parkinsonism and Parkinson's disease. Neuroscience Letters211:13-16]. It has also been shown that IL-1β inhibits long-termpotentiation in the hippocampus [Murray C A, Lynch M A (1998). Evidencethat increase hippocampal expression of the cytokine interleukin-1β is acommon trigger for age and stress-induced impairments in long-termpotentiation. Journal of Neuroscience 18:2974-2981]. Long-termpotentiation in the hippocampus is a form of synaptic plasticity and isgenerally considered to be an appropriate model for memory and learning[Bliss T V P, Collinridge G L, (1993). A synaptic model of memory:long-term potentiation in the hippocampus, Nature 361:31-39]. Thus,inappropriate cytokine expression in the brain is currently believed tobe involved in the development and progression of neurodegenerativediseases. Consequently, the finding of success in CHS treatment reportedin the above Examples, with its attendant down-regulation of Th-1inflammatory cytokines, is indicative of successful use of the processand compositions in the treatment and prophylaxis of a wide variety ofneurological disorders including those discussed above.

1. The use of apoptotic bodies and/or apoptotic cells in treatmentand/or prophylaxis in mammalian patients of neurodegenerative and otherneurological medical disorders.
 2. The use of apoptotic bodies and/orapoptotic cells in the preparation of a medicament for the treatmentand/or prophylaxis of neurodegenerative and other neurological medicaldisorders in mammalian patients.
 3. The use of claim 2 wherein apoptoticbodies and/or apoptotic cells are in a liquid suspension along withviable cells.
 4. The use of claim 3 wherein the apoptotic bodies and/orapoptotic cells comprise from 10% to 90% of the cellular portion of thesuspension.
 5. The use of claim 4 wherein the apoptotic bodies and/orapoptotic cells comprise from 30% to 70% of the cellular portion of thesuspension.
 6. The use of claim 1 or 2 wherein the apoptotic bodiesand/or cells are derived from extracorporeal treatment of blood cellscompatible with those of the mammalian patient.
 7. The use of claim 1 or2 wherein the apoptotic bodies and/or cells are derived from establishedcultured cell lines.
 8. The use of claim 6 wherein the blood cells arewhite blood cells of blood compatible with that of the mammalianpatient.
 9. The use of claim 8 wherein the blood cells are the patient'sown white blood cells.
 10. The use of claim 9 wherein the blood cellsare the patient's own T lymphocytes.
 11. The use of claim 1 or 2 whereinthe disorder is selected from the group consisting of Alzheimer'sdisease, senile dementia, multiple sclerosis, depression, Down'ssyndrome, Huntington's disease, peripheral neuropathies, spinal corddiseases, neuropathic joint diseases, chronic inflammatory demyelinatingdisease (CIPD), neuropathies including mononeuropathy, polyneuropathy,symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscularjunction disorders, myasthenias and Parkinson's disease.
 12. The use ofclaim 1 or 2 further comprising administering to a human patient adosage of apoptotic bodies and/or apoptotic cells comprising from 10,000to 10,000,000 apoptotic bodies and/or apoptotic cells per kilogram bodyweight of the patient.
 13. The use of claim 12 wherein the dosagecontains from 500,000 to 5,000,000 apoptotic bodies and/or apoptoticcells per kilogram body weight of the patient.
 14. The use of claim 12wherein the dosage contains from 1,500,000 to 4,000,000 apoptotic bodiesand/or apoptotic cells per kilogram body weight of the patient.
 15. Amethod for treatment of or prophylaxis against T-cell-mediated andinflammatory disorders in a mammalian patient, which comprisesadministering to the patient an effective amount of apoptotic bodiesand/or apoptotic cells.
 16. A pharmaceutical composition comprising apharmaceutically acceptable excipient and an effective amount ofapoptotic bodies and/or apoptotic cells.
 17. The pharmaceuticalcomposition of claim 16 which is suitable for administration to amammalian patient to treat or to effect prophylaxis againstneurodegenerative and other neurological medical disorders.
 18. Thecomposition of claim 16 or 17 comprising a liquid suspension of cellularmaterial, from 10% to 90% of the cellular material being apoptoticbodies and/or apoptotic cells.
 19. A unit dosage composition foradministration to a human patient for alleviation or prophylaxis of aneurological or neurodegenerative disorder, comprising a liquidsuspension of cellular material including from about 10,000 to10,000,000 apoptotic cells and/or apoptotic bodies per kilogram ofpatient body weight.